Transgenic mice technology involves the introduction of new or altered genetic material into the mouse germ line. This results in lineages that carry the new integrated genetic material.
Endoplasmic reticulum (ER) plays an important role in many functions of the cell. ER is not only the protein folding and processing machinery of the cell but it plays an important role in Ca2+ storage and regulation of intracellular Ca2+ homeostasis (Pozzan et al., 1994). It is also important in gene regulation (unfolded protein response) (Tirasophon et al., 1998; Welihinda et al., 1997). There are a number of ER resident proteins (including CRT) which are essential for the proper implementation of these functions.
Studies on tumor angiogenesis have resulted in a significant progress in our understanding of the genetic and molecular mechanisms that control the development of the vascular system. The embryonic origin of the vascular system is best understood with respect to endothelial cells (Cleaver and Krieg, 1999). These cells are the defining cell types of the vascular system. During embryogenesis, these cells differentiate (from angioblast origin), migrate, and assemble into the vascular network. Subsequently pericytes are recruited to the periphery of the endothelium and differentiate into vascular smooth muscle cells (Manasek, 1971).
The formation of the vascular system is achieved by the coordination of two processes, namely vasculogenesis and angiogenesis (Risau and Lemmon, 1988), (Pardanaud et al., 1989). Vasculogenesis is the first step in vascular development leading to layout of the initial primitive vascular network, the capillary plexus (Risau and Flamme, 1995). Angiogenesis is a later process, which involves the sprouting, branching and differential growth of blood vessels to form the mature vessel (Risau and Flamme, 1995). There are two types of angiogenesis: sprouting angiogenesis, involving true sprouting of capillaries from preexisting blood vessels, and non-sprouting angiogenesis (or intussusception) which involves the splitting of preexisting vessels (Folkman and Klagsbrun, 1987; Klagsbrun and D'Amore, 1991; Patan et al., 1996). Vascularization of tissues like the yolk sac, embryonic brain, kidney, thymus, limb bud and intersomitic vessels are formed by sprouting angiogenesis (Ekblom et al., 1982; Le Lievre and Le Douarin, 1975; Bar, 1980; Joterau and Le Douarin, 1978; Stewart and Wiley, 1981; Coffin and Poole, 1988). However, in the developing lung, myocardium, chorioallantoic membrane, endothelial wound healing and the development of coronary arteries, vascularization occurs by non-sprouting, or intussusception angiogenesis (Flamme and Risau, 1992; Burri and Tarek, 1990; van Groningen et al., 1991; Patan et al., 1993; Patan et al., 1996; Reidy and Schwartz, 1981; Bogers et al., 1989).
A large number of molecules can affect angiogenesis and vasculogenesis, including a number of growth factors, their receptors, and components of the extracellular matrix (ECM) (reviewed in Cleaver and Krieg, 1999). The receptor tyrosine kinases expressed on the surface of endothelial cells also play important roles in initiating the program of endothelial cell growth during development and subsequent vascularization during wound healing and tumorigenesis. VEGF and its receptor Flk-1 are thought to be responsible for both primary vessel formations during vasculogenesis and angiogenic invasion of the developing organs (Flamme et al., 1995; Cleaver et al., 1997; Cleaver and Krieg, 1999). Both Flk-1 and Flt-1 (placental growth factor receptor) are expressed exclusively in the endothelial cells (Flamme et al., 1995; Peters et al., 1993). Tie-2 is another receptor tyrosine kinase which is highly expressed in the endothelial cells during embryogenesis and adult life (Davis et al., 1996; Suri et al., 1996). This receptor and its ligands, angiopoietin-1 and 2, are involved in angiogenesis and later vascular remodeling (Dumont et al., 1995; Sato et al., 1995). Targeted disruption of the Tie-2 gene in mice resulted in embryonic lethality due to defects in the integrity of the endothelium, and consequently defects in cardiac and vascular development (Dumont et al., 1992; Dumont et al., 1994; Dumont et al., 1995). These observations demonstrate that the Tie-2 signaling pathway plays a critical role in the differentiation, proliferation, and survival of endothelial cells as well as heart development in the mouse embryo (Dumont et al., 1994; Sato et al., 1995). Because gene targeted deletion of CRT results in similar cardiovascular defects in the embryos (Mesaeli et al., 1999), one may hypothesize that CRT plays a role in the growth factor signaling pathway. Indeed, an inverse relationship between expression of CRT and total cellular tyrosine phosphorylation level has been reported in cultured cells (Fadel et al., 1999) suggesting that the protein may affect tyrosine phosphorylation-dependent signaling.
The ECM and cell adhesion proteins can also regulate the process of vasculogenesis and angiogenesis by modulating growth, differentiation and migration of the endothelial cells (Risau and Lemmon, 1988; Ausprunk et al., 1991). For example, fibronectin is essential for the assembly of the vessel (Risau and Lemmon, 1988; Hynes, 1990), while collagen and laminin become important in later stages of vessel development (Hynes, 1990). Blocking of α5β1 integrin function results in major defects in early vasculogenesis (Drake et al., 1992; Yang et al., 1993). Blocking the β3 family of integrins results in defects in angiogenesis and vascular cell survival (Brooks et al., 1994a; Brooks et al., 1994b). CRT may influence vessel formation through regulating the expression and function of cell adhesion proteins. For example differential expression of CRT affects integrin function (Leung-Hagesteijn et al., 1994; Coppolino et al., 1997). In addition, overexpression of CRT results in up-regulation of vinculin and N-cadherin (Fadel et al., 1999; Opas et al., 1996). It is still presently unclear if CRT alters angiogenesis through an effect on ECM or cell adhesion proteins or both.